Dendritic cells (DC) were shown to enhance the efficiency of HIV-1 infection of T cells through the binding of viral gp120 to a DC surface C-type lectin-like receptor, DC-SIGN, and subsequent delivery of the viral particles to T cells. The recruitment of DC-SIGN by HIV to facilitate the viral infection makes the receptor a potential new target for vaccines and anti-viral therapy. We have expressed several soluble forms of DC-SIGN and DC-SIGNR using a bacterial system followed by in vitro reconstitution. The refolded CRD, R8 and Ecto forms of DC-SIGN exist in solution as monomers, dimers and octamers, respectively, suggesting that the function of the extracellular repeat region is to stabilize the oligomeric form of the receptor. Furthermore, the gp120 binding affinity of DC-SIGN increases as the receptor oligomerizes, suggesting that oligomerization plays an important role in the receptor recognition of gp120. We have also crystallized the R8 construct of DC-SIGNR. The structure was solved to 1.5 angstrom resolution. The structure shows that the CRD domain adopts a typical C-type lectin fold with both calcium and carbohydrate binding sites visible in the electron densities. The construct contains a single repeat and it assumes a helical conformation. Antibodies were raised against the recombinant DC-SIGN(R8) and one (II.1) exhibits near complete inhibition of the receptor/ gp120 binding both in solution and on the surface of immature dendritic cells, whereas the others display variable inhibitory effects. In addition to the anti-DC-SIGN antibodies, two anti-gp120 antisera were also tested but were not able to block the gp120 binding to DC-SIGN. All constructs of the soluble receptor were subjected to crystallization screening experiments and the preliminary results show small crystals of the receptor can be obtained. To address the issue of whether DC-SIGN binding to gp120 leads to surface expression of HIV or antigen processing, we measured the release of gp120 under an endosomal pH. Earlier published results suggest that gp120 is retained by DC-SIGN in early endosomes. When measuring the pH dependent gp120 binding by DC-SIGN, our results showed that the gp120 affinity was reduced in a low pH environment, making it unlikely that the receptor retains the HIV viron through a DC-SIGN and gp120 interaction. Therefore, following internalization, gp120 is most likely to be released to the degradatioin pathway for antigen processing and presentation. In contrast to gp120 binding, DC-SIGN and DC-SIGNR bound to ICAM molecules with micromolar affinities, which were independent of the receptor oligomerization state. Furthermore, DC-SIGNR recognized ICAM-3 with a similar affinity as it did other cell surface glycoproteins such as the type II and III Fcgamma receptors. This suggests that the DC-SIGN and ICAM recognition is likely non-specific and that ICAM molecules may not be the physiological ligand of DC-SIGN. In a separate initiative, we began to examine the role of Siglec, a class of immunoglobulin-like carbohydrate-binding lectins, in HIV-1 host infection. In particular, macrophages are known to express Siglec-1 and -3. We found that, in addition to Siglec-1, macrophages also express Siglec-5 and -7. Preliminary results show that R5 trophic HIV are subject to inhibition by sialyllactose, an inhibitor of Siglecs. Furthermore, soluble Siglec-1-Fc protein also partially inhibits the R5 virus entry. These results suggest that Siglecs may function as an auxillary receptor for HIV infection.